BTLA agonist antibodies and uses thereof

ABSTRACT

Antibodies which bind BTLA, and methods of using same, are provided, said antibodies are useful as agents for treating conditions associated with autoimmune disease including treating lupus.

The present invention is in the field of medicine. More particularly,the present invention relates to antibodies directed against B and TLymphocyte Attenuator (BTLA) and pharmaceutical compositions thereof.The antibodies of the present invention are expected to be useful in thetreatment of autoimmune diseases such as lupus.

Lupus is an autoimmune disease with heterogeneous features, includingskin, oral, muscle & joint, cardiac, peripheral blood, lung, kidney,reproductive, and CNS manifestations. Lupus patients are at risk forserious and life-threatening cardiovascular, renal and neuropsychiatricdisease. The standard of care includes numerous steroids, which havemany unfavorable and/or dangerous side effects. There is a need fortherapies to manage disease and allowing for reduction or elimination ofsteroid use.

B and T Lymphocyte Attenuator (BTLA; CD272) is an Ig superfamily memberand part of a family of checkpoint receptors that negatively regulateimmune cell activation. BTLA is primarily expressed on B cells, T cells,and dendritic cells. The natural ligand for BTLA is the TNF receptorsuperfamily member, herpes virus entry mediator (HVEM; TNFRSF14).

Human HVEM-Fc has been reported to bind to human BTLA expressed in 293Tcells with a K_(D) of 112 nM as detected by flow cytometry. (Cheung etal., PNAS, Sep. 13, 2005, 102:37; 13218-13223). Binding of HVEM to BTLAleads to tyrosine-phosphorylation of two conserved immunoreceptortyrosine-based inhibitory motif domains on the cytoplasmic domain ofBTLA. This phosphorylation leads to recruitment of, via two Src homology2 domains, protein tyrosine phosphatases that impart the inhibitoryactivity of BTLA by dephosphorylating and down-regulating positive cellreceptor signaling (eg. T cell receptor or B cell receptor signaltransduction cascades), thus leading to suppression of immune cellactivation. In a mouse model prone to spontaneously develop lupus-likediseases (MRL-lpr mice), BTLA-deficient mice have more severelymphocytic infiltration in salivary glands, lungs, pancreas, kidneysand joints compared to BTLA-expressing mice. Therefore, BTLA agonistantibodies may provide a benefit for patients having autoimmune diseasessuch as lupus.

Agonist antibodies to BTLA are known in the art. For example, U.S. Pat.No. 8,563,694 (the '694 patent) discloses BTLA agonist antibodies thateither block (Mab21H6 and Mab19A7) or do not block (Mab8D5 and Mab8A3)HVEM binding to BTLA. The '694 patent describes an ongoing need todevelop treatments that exploit the inhibitory role of BTLA inlymphocyte responses, while allowing for BTLA-HVEM binding. However,there is a lack of BTLA agonist antibodies that mimic the binding ofHVEM to BTLA for the treatment of autoimmune diseases. An antibody“mimics” HVEM binding to BTLA if the antibody has an epitope thatsignificantly overlaps the binding site of HVEM, and there is structuralsimilarity between the antibody and HVEM. There is also a lack of BTLAagonist antibodies that bind human BTLA and are useful to study in invivo pre-clinical models of autoimmune diseases such as murine andcynomolgus monkey models. Thus, there remains a need for alternativeBTLA agonist antibodies.

The antibodies of the present invention seek to provide alternative BTLAagonist antibodies. Such BTLA agonist antibodies may be useful in thetreatment of autoimmune diseases such as lupus. Such BTLA agonistantibodies are able to bind BTLA from multiple species such as human,cynomolgus monkey, and/or murine BTLA. In addition, such BTLA agonistantibodies demonstrate increased in vitro activity compared to anantibody having the same heavy chain variable region and light chainvariable region as Mab8D5. The antibodies of the present inventionspossess at least one of these desirable characteristics.

One such BTLA agonist antibody is able to bind human, cynomolgus monkey,and murine BTLA. Surprisingly, this antibody has this desiredcross-reactivity because it mimics HVEM binding to BTLA. This antibodyalso has a higher binding affinity to BTLA as compared to HVEM bindingBTLA. This may provide a benefit for patients having disease states withtransient levels of HVEM, wherein it may desirable to have aBTLA-mimicking agonist antibody on-board during times when the patienthas a reduction in HVEM.

The present inventions provide antibodies that bind to BTLA and activateand/or enhance BTLA-mediated signaling (BTLA agonist antibodies). Thepresent inventions provide an antibody that comprises a light chainvariable region (LCVR) and a heavy chain variable region (HCVR), whereinthe LCVR comprises complementarity determining regions (CDRs) LCDR1,LCDR2, and LCDR3 and the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3,and wherein the amino acid sequence of LCDR1 is SEQ ID NO: 22, the aminoacid sequence of LCDR2 is SEQ ID NO: 25, the amino acid sequence ofLCDR3 is SEQ ID NO: 28, the amino acid sequence of HCDR1 is SEQ ID NO:13, the amino acid sequence of HCDR2 is SEQ ID NO: 16, and the aminoacid sequence of HCDR3 is SEQ ID NO: 19. In an embodiment, the antibodycomprises a LCVR and a HCVR, and wherein the amino acid sequence of theLCVR is SEQ ID NO: 4, and the amino acid sequence of the HCVR is SEQ IDNO: 3. In another embodiment, the antibody comprises a light chain (LC)and a heavy chain (HC), and wherein the amino acid sequence of the LC isSEQ ID NO: 2, and the amino acid sequence of the HC is SEQ ID NO: 1. Inyet another embodiment, the antibody comprises 2 LCs and 2 HCs, whereinthe amino acid sequence of each LC is SEQ ID NO: 2, and the amino acidsequence of each HC is SEQ ID NO: 1.

The present inventions also provide a BTLA agonist antibody wherein theamino acid sequence of LCDR1 is SEQ ID NO: 23, the amino acid sequenceof LCDR2 is SEQ ID NO: 26, the amino acid sequence of LCDR3 is SEQ IDNO: 29, the amino acid sequence of HCDR1 is SEQ ID NO: 14, the aminoacid sequence of HCDR2 is SEQ ID NO: 17, and the amino acid sequence ofHCDR3 is SEQ ID NO: 20. In an embodiment, the amino acid sequence of theLCVR is SEQ ID NO: 8, and the amino acid sequence of the HCVR is SEQ IDNO: 7. In another embodiment, the amino acid sequence of the LC is SEQID NO: 6, and the amino acid sequence of the HC is SEQ ID NO: 5. In yetanother embodiment, the antibody comprises 2 LCs and 2 HCs, wherein theamino acid sequence of each LC is SEQ ID NO: 6, and the amino acidsequence of each HC is SEQ ID NO: 5.

The present inventions also provide a BTLA agonist antibody wherein theamino acid sequence of LCDR1 is SEQ ID NO: 24, the amino acid sequenceof LCDR2 is SEQ ID NO: 27, the amino acid sequence of LCDR3 is SEQ IDNO: 30, the amino acid sequence of HCDR1 is SEQ ID NO: 15, the aminoacid sequence of HCDR2 is SEQ ID NO: 18, and the amino acid sequence ofHCDR3 is SEQ ID NO: 21. In an embodiment, the amino acid sequence of theLCVR is SEQ ID NO: 12, and the amino acid sequence of the HCVR is SEQ IDNO: 11. In another embodiment, the amino acid sequence of the LC is SEQID NO: 10, and the amino acid sequence of the HC is SEQ ID NO: 9. In yetanother embodiment, the antibody comprises 2 LCs and 2 HCs, wherein theamino acid sequence of each LC is SEQ ID NO: 10, and the amino acidsequence of each HC is SEQ ID NO: 9.

The present invention also provides an antibody that binds BTLA, whereinthe antibody is generated by steps comprising immunizing rabbits withFc-tagged extracellular domain (ECD) domain of human BTLA and boostingwith human and mouse BTLA-Fc tagged proteins. The amino acid sequence ofthe human BTLA ECD is amino acids 31-150 of SEQ ID NO: 31.

The present invention provides a BTLA agonist antibody that mimics HVEMbinding to BTLA. The present invention also provides a BTLA agonistantibody that is able to bind human, cynomulgus monkey, and murine BTLA.

The present invention also provides a pharmaceutical compositioncomprising an antibody of the present invention, and one or morepharmaceutically acceptable carriers, diluents, or excipients. In someembodiments, pharmaceutical compositions of the present invention can beused in the treatment of one or more of rheumatic, neural, anddermatology disease, whereby such treatment comprises administering to apatient in need thereof an effective amount of a pharmaceuticalcomposition of the present invention. In some particular embodiments,the rheumatic disease is at least one of lupus nephritis, systemic lupuserythematosus, and rheumatoid arthritis. In other particularembodiments, the dermatology disease is at least one of atopicdermatitis and psoriasis. In other particular embodiments, the neuraldisease is multiple sclerosis.

The present invention also provides a method of treating a patienthaving one or more of rheumatic, neural, and dermatology disease,comprising administering to a patient in need thereof an effectiveamount of an antibody of the present invention. In some suchembodiments, the rheumatic disease is at least one of lupus nephritis,systemic lupus erythematosus, and rheumatoid arthritis. In otherparticular embodiments, the dermatology disease is at least one ofatopic dermatitis and psoriasis. In other particular embodiments, theneural disease is multiple sclerosis.

The present invention also provides an antibody of the present inventionor pharmaceutical composition thereof for use in therapy. In someembodiments, the present invention provides an antibody of the presentinvention or pharmaceutical composition thereof for use in the treatmentof one or more of rheumatic, neural, and dermatology disease. In somesuch embodiments, the rheumatic disease is at least one of lupusnephritis, systemic lupus erythematosus, and rheumatoid arthritis. Inother particular embodiments, the dermatology disease is at least one ofatopic dermatitis and psoriasis. In other particular embodiments, theneural disease is multiple sclerosis.

The present invention also provides the use of an antibody of thepresent invention or a pharmaceutical composition thereof in themanufacture of a medicament for the treatment of one or more ofrheumatic, neural, and dermatology disease. In some such embodiments,the rheumatic disease is at least one of lupus nephritis, systemic lupuserythematosus, and rheumatoid arthritis. In other particularembodiments, the dermatology disease is at least one of atopicdermatitis and psoriasis. In other particular embodiments, the neuraldisease is multiple sclerosis.

The present invention provides a DNA molecule comprising apolynucleotide sequence encoding a polypeptide having the amino acidsequence of SEQ ID NO:1, SEQ ID NO:5, or SEQ ID NO:9. The presentinvention also provides a DNA molecule comprising a polynucleotidesequence encoding a polypeptide having the amino acid sequence of SEQ IDNO:2, SEQ ID NO:6, or SEQ ID NO:10.

The present invention provides a DNA molecule comprising apolynucleotide sequence encoding a polypeptide having the amino acidsequence of SEQ ID NO: 1, and comprising a polynucleotide sequenceencoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.The present invention also provides a DNA molecule comprising apolynucleotide sequence encoding a polypeptide having the amino acidsequence of SEQ ID NO: 1, and a DNA molecule comprising a polynucleotidesequence encoding a polypeptide having the amino acid sequence of SEQ IDNO: 2. In a particular embodiment the polynucleotide sequence encoding apolypeptide having the amino acid sequence of SEQ ID NO: 1 is SEQ ID NO:35 and the polynucleotide sequence encoding a polypeptide having theamino acid sequence of SEQ ID NO: 2 is SEQ ID NO: 36.

The present invention also provides a DNA molecule comprising apolynucleotide sequence encoding a polypeptide having the amino acidsequence of SEQ ID NO: 5, and comprising a polynucleotide sequenceencoding a polypeptide having the amino acid sequence of SEQ ID NO: 6.The present invention also provides a DNA molecule comprising apolynucleotide sequence encoding a polypeptide having the amino acidsequence of SEQ ID NO: 5, and a DNA molecule comprising a polynucleotidesequence encoding a polypeptide having the amino acid sequence of SEQ IDNO: 6. In a particular embodiment the polynucleotide sequence encoding apolypeptide having the amino acid sequence of SEQ ID NO: 5 is SEQ ID NO:37, the polynucleotide sequence encoding a polypeptide having the aminoacid sequence of SEQ ID NO: 6 is SEQ ID NO: 38.

The present invention also provides a DNA molecule comprising apolynucleotide sequence encoding a polypeptide having the amino acidsequence of SEQ ID NO: 9, and comprising a polynucleotide sequenceencoding a polypeptide having the amino acid sequence of SEQ ID NO: 10.The present invention also provides a DNA molecule comprising apolynucleotide sequence encoding a polypeptide having the amino acidsequence of SEQ ID NO: 9, and a DNA molecule comprising a polynucleotidesequence encoding a polypeptide having the amino acid sequence of SEQ IDNO: 10. In a particular embodiment the polynucleotide sequence encodinga polypeptide having the amino acid sequence of SEQ ID NO: 9 is SEQ IDNO: 39, the polynucleotide sequence encoding a polypeptide having theamino acid sequence of SEQ ID NO: 10 is SEQ ID NO: 40.

Further, the present invention provides a mammalian cell comprising aDNA molecule comprising a polynucleotide sequence encoding a polypeptidehaving the amino acid sequence of SEQ ID NO: 1 and a DNA moleculecomprising a polynucleotide sequence encoding a polypeptide having theamino acid sequence of SEQ ID NO: 2. The present invention also providesa mammalian cell comprising a DNA molecule comprising a polynucleotidesequence encoding a polypeptide having the amino acid sequence of SEQ IDNO: 5 and a polypeptide having the amino acid sequence of SEQ ID NO: 6.The present invention also provides a mammalian cell comprising a DNAmolecule comprising a polynucleotide sequence encoding a polypeptidehaving the amino acid sequence of SEQ ID NO: 9 and a polypeptide havingthe amino acid sequence of SEQ ID NO: 10. In an embodiment the mammaliancell line is a Chinese Hamster Ovary (CHO) or Hamster embryonic kidney(HEK) cell line.

The present invention also provides a mammalian cell comprising a DNAmolecule comprising a polynucleotide sequence encoding a polypeptidehaving the amino acid sequence of SEQ ID NO:1 and/or a DNA moleculecomprising a polynucleotide sequence encoding a polypeptide having theamino acid sequence of SEQ ID NO: 2, wherein the cell is capable ofexpressing an antibody comprising a HC having the amino acid sequence ofSEQ ID NO:1 and a LC having the amino acid sequence of SEQ ID NO: 2.Preferably the mammalian cell comprises a DNA molecule comprising apolynucleotide sequence encoding a polypeptide having the amino acidsequence of SEQ ID NO:1 and a polypeptide having the amino acid sequenceSEQ ID NO: 2. In an embodiment the mammalian cell line is a CHO or HEKcell line.

The present invention also provides a mammalian cell comprising a DNAmolecule comprising a polynucleotide sequence encoding a polypeptidehaving the amino acid sequence of SEQ ID NO:5 and/or a DNA moleculecomprising a polynucleotide sequence encoding a polypeptide having theamino acid sequence of SEQ ID NO: 6, wherein the cell is capable ofexpressing an antibody comprising a HC having the amino acid sequence ofSEQ ID NO:5 and a LC having the amino acid sequence of SEQ ID NO: 6.Preferably the mammalian cell comprises a DNA molecule comprising apolynucleotide sequence encoding a polypeptide having the amino acidsequence of SEQ ID NO:5 and a polypeptide having the amino acid sequenceSEQ ID NO: 6. In an embodiment the mammalian cell line is a CHO or HEKcell line.

The present invention also provides a mammalian cell comprising a DNAmolecule comprising a polynucleotide sequence encoding a polypeptidehaving the amino acid sequence of SEQ ID NO:9 and/or a DNA moleculecomprising a polynucleotide sequence encoding a polypeptide having theamino acid sequence of SEQ ID NO: 10, wherein the cell is capable ofexpressing an antibody comprising a HC having the amino acid sequence ofSEQ ID NO:9 and a LC having the amino acid sequence of SEQ ID NO: 10.Preferably the mammalian cell comprises a DNA molecule comprising apolynucleotide sequence encoding a polypeptide having the amino acidsequence of SEQ ID NO:9 and a polypeptide having the amino acid sequenceSEQ ID NO: 10. In an embodiment the mammalian cell line is a CHO or HEKcell line.

In another embodiment, the present invention provides a process forproducing an antibody comprising a LC having an amino acid sequence ofSEQ ID NO: 2 and a HC having an amino acid sequence of SEQ ID NO: 1,wherein the process comprises cultivating a mammalian cell comprising aDNA encoding a LC having an amino acid sequence of SEQ ID NO: 2 and/or aHC having an amino acid sequence of SEQ ID NO: 1 under conditions suchthat the antibody is expressed, and recovering the expressed antibody.The invention includes an antibody obtainable by the process of theinvention as described immediately above.

The present invention also provides a process for producing an antibodycomprising a LC having an amino acid sequence of SEQ ID NO: 6 and a HChaving an amino acid sequence of SEQ ID NO: 5, wherein the processcomprises cultivating a mammalian cell comprising a DNA encoding a LChaving an amino acid sequence of SEQ ID NO: 6 and/or a HC having anamino acid sequence of SEQ ID NO: 5 under conditions such that theantibody is expressed, and recovering the expressed antibody. Theinvention includes an antibody obtainable by the process of theinvention as described immediately above.

The present invention also provides a process for producing an antibodycomprising a LC having an amino acid sequence of SEQ ID NO: 10 and a HChaving an amino acid sequence of SEQ ID NO: 9, wherein the processcomprises cultivating a mammalian cell comprising a DNA encoding a LChaving an amino acid sequence of SEQ ID NO: 10 and/or a HC having anamino acid sequence of SEQ ID NO: 9 under conditions such that theantibody is expressed, and recovering the expressed antibody. Theinvention includes an antibody obtainable by the process of theinvention as described immediately above.

The present invention includes a process for producing an antibody,which antibody comprises two HCs and two LCs, in which the aminosequence of each of the two HCs is SEQ ID NO: 1, and the amino acidsequence of each of the two LCs is SEQ ID NO: 2, and which processcomprises: a) cultivating a mammalian cell of the invention, asdescribed above, under conditions such that the antibody is expressed,and b) recovering the expressed antibody. The invention includes anantibody obtainable by the process of the invention as describedimmediately above.

The present invention also includes a process for producing an antibody,which antibody comprises two HCs and two LCs, in which the aminosequence of each of the two HCs is SEQ ID NO: 5 and the amino acidsequence of each of the two LCs is SEQ ID NO: 6, and which processcomprises: a) cultivating a mammalian cell of the invention, asdescribed above, under conditions such that the antibody is expressed,and b) recovering the expressed antibody. The invention includes anantibody obtainable by the process of the invention as describedimmediately above.

The present invention also includes a process for producing an antibody,which antibody comprises two HCs and two LCs, in which the aminosequence of each of the two HCs is SEQ ID NO: 9 and the amino acidsequence of each of the two LCs is SEQ ID NO: 10, and which processcomprises: a) cultivating a mammalian cell of the invention, asdescribed above, under conditions such that the antibody is expressed,and b) recovering the expressed antibody. The invention includes anantibody obtainable by the process of the invention as describedimmediately above.

The present invention provides an antibody that contacts human BTLA at astructural and functional epitope having the following residues of SEQID NO: 31: Arg at position 42 and His at position 127. The presentinvention also provides an antibody that contacts human BTLA at astructural and functional epitope comprising Arg at position 42 of theamino acid sequence given by SEQ ID NO: 31.

The present invention provides an antibody that contacts human BTLA at anovel structural epitope having the following residues of SEQ ID NO: 31:Asp at position 35, Gln at position 37, Arg at position 42, Leu atposition 74, Gly at position 76, Cys at position 79, Arg at position114, Phe at position 119, Gln at position 120, Asn at position 122, Serat position 128. In a preferred embodiment, the antibody 22B3 is said tomimic HVEM binding to BTLA because the HCDR3 of antibody 22B3 isstructurally similar to HVEM. Preferably, when the BTLA:antibody crystalstructure is aligned with the BTLA:HVEM crystal structure in a programsuch as PyMOL™, an antibody CDR loop adopts a conformation similar tothe HVEM loop comprising amino acid residues 69 to 72 (amino acids ELTGof SEQ ID NO:41).

The present invention provides an antibody that contacts human BTLA at afunctional epitope having Asp at position 52 of SEQ ID NO:31. Theantibody contacts a novel structural epitope having the followingresidues of SEQ ID NO: 31: His at position 46, Glu at position 55, Gluat position 103, Pro at position 104, Leu at position 106, Pro atposition 107, Thr at position 134, Ala at position 139.

The present invention provides an antibody that contacts human BTLA at afunctional epitope having His at position 68 and Lys at position 81 ofSEQ ID NO:31. In an embodiment, the antibody contacts a novel structuralepitope having the following residues of SEQ ID NO: 31: Tyr at position62, Ala at position 64, His at position 68, Arg at position 85, Glu atposition 91, Phe at position 98, Asn at position 118.

The present invention provides an antibody that contacts human BTLA at anovel structural epitope having the following residues of SEQ ID NO: 31:Asp at position 35, Gln at position 37, Arg at position 42, Leu atposition 74, Gly at position 76, Cys at position 79, Arg at position114, Phe at position 119, Gln at position 120, Asn at position 122, andIle at position 124, Ser at position 128.

As used herein, an “antibody” is an immunoglobulin molecule comprising 2HCs and 2 LCs interconnected by disulfide bonds. The amino terminalportion of each LC and HC includes a variable region of about 100-120amino acids primarily responsible for antigen recognition via the CDRscontained therein. The CDRs are interspersed with regions that are moreconserved, termed framework regions (“FR”). Each LCVR and HCVR iscomposed of 3 CDRs and 4 FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The 3 CDRs of the LC are referred to as “LCDR1, LCDR2, andLCDR3,” and the 3 CDRs of the HC are referred to as “HCDR1, HCDR2, andHCDR3.” The CDRs contain most of the residues which form specificinteractions with the antigen. That is, the CDRs contain most of theresidues that are in contact with (within 4.5 Å) the antigen's residues.The functional ability of an antibody to bind a particular antigen is,thus, largely influenced by the amino acid residues within the six CDRs.Assignment of amino acids to CDR domains within the LCVR and HCVRregions of the antibodies of the present invention is based on thewell-known Kabat numbering convention (Kabat, et al., Ann. NY Acad. Sci.190:382-93 (1971); Kabat et al., Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242 (1991)), and Chothia (Chothia C, Lesk A M.Canonical structures for the hypervariable regions of immunoglobulins.J. Mol. Biol. 1987; 196:901-17. Chothia C, Lesk A M, Tramontano A,Levitt M, Smith-Gill S J, Air G, Sheriff S, Padlan E A, Davies D, TulipW R, et al. Conformations of immunoglobulin hypervariable regions.Nature. 1989; 342:877-83). The starting amino acid residue of HCDR1 isdefined by Chothia and the ending amino acid reside for HCDR1 is definedby Kabat. The starting and ending amino acid residues for HCDR2, HCDR3,LCDR1, LCDR2, and LCDR3 are defined by Kabat.

The term “epitope” as used herein may refer to a structural epitope(sites of an antigen that are in contact with the variable region of anantibody) and/or a functional epitope (sites of an antigen that may ormay not be in contact with the variable region of an antibody and arenecessary for antibody binding). The structural epitope is determined byX-ray crystallography wherein any residue on human BTLA within 4.5A ofanother residue on the bound Fab is considered to be a contact site.

The antibodies of the present invention may be prepared and purifiedusing known methods. For example, cDNA sequences encoding a HC (forexample the amino acid sequence given by SEQ ID NO: 1) and a LC (forexample, the amino acid sequence given by SEQ ID NO: 2) may be clonedand engineered into a GS (glutamine synthetase) expression vector. Theengineered immunoglobulin expression vector may then be stablytransfected into CHO cells. As one skilled in the art will appreciate,mammalian expression of antibodies will result in glycosylation,typically at highly conserved N-glycosylation sites in the Fc region.Stable clones may be verified for expression of an antibody specificallybinding to BTLA. Positive clones may be expanded into serum-free culturemedium for antibody production in bioreactors. Medium, into which anantibody has been secreted, may be purified by conventional techniques.For example, the medium may be conveniently applied to a Protein A or GSepharose FF column that has been equilibrated with a compatible buffer,such as phosphate buffered saline. The column is washed to removenonspecific binding components. The bound antibody is eluted, forexample, by pH gradient and antibody fractions are detected, such as bySDS-PAGE, and then pooled. The antibody may be concentrated and/orsterile filtered using common techniques. Soluble aggregate andmultimers may be effectively removed by common techniques, includingsize exclusion, hydrophobic interaction, ion exchange, or hydroxyapatitechromatography. The product may be immediately frozen, for example at−70° C., or may be lyophilized.

An antibody of the present invention can be incorporated into apharmaceutical composition which can be prepared by methods well knownin the art and comprise an antibody of the present invention and one ormore pharmaceutically acceptable carriers, diluents, or excipients.

A pharmaceutical composition comprising an effective amount of anantibody of the present invention can be administered to a patient atrisk for, or exhibiting, diseases or disorders as described herein byparental routes (e.g., subcutaneous, intravenous, intraperitoneal,intramuscular, or transdermal). An “effective amount” refers to anamount necessary (at dosages and for periods of time and for the meansof administration) to achieve the desired therapeutic result. Aneffective amount of the antibody may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the antibody to elicit a desired response in the individual.An effective amount is also one in which any toxic or detrimentaleffects of the antibody of the present invention are outweighed by thetherapeutically beneficial effects.

The antibodies of the present invention can be used in the treatment ofpatients. More particularly the antibodies of the present invention areexpected to treat one or more of rheumatic, neural, and dermatologydisease. Rheumatic diseases are characterized by inflammation that canaffect a person's joints, muscles, and/or organs. One such rheumaticdisease is systemic lupus erythematosus (SLE).

As used interchangeably herein, “treatment” and/or “treating” and/or“treat” are intended to refer to all processes wherein there may be aslowing, interrupting, arresting, controlling, stopping, or reversing ofthe progression of the disorders described herein, but does notnecessarily indicate a total elimination of all disorder symptoms.Treatment includes administration of an antibody of the presentinvention for treatment of a disease or condition in a human that wouldbenefit from an increase in BTLA activity, and includes: (a) inhibitingfurther progression of the disease; and (b) relieving the disease, i.e.,causing regression of the disease or disorder or alleviating symptoms orcomplications thereof.

Antibody Engineering

The antibodies of the present invention were generated by immunizingrabbits with Fc-tagged extracellular domain (ECD) domain of human BTLAand boosting with mouse BTLA-Fc tagged protein (25F7) or alternatelywith human and mouse BTLA-Fc tagged proteins (22B3 and 23C8). Screeningwas done with histidine-tagged human, mouse, and cynomolgus monkey BTLAto identify cross reactivity. The amino acid sequence of human BTLA isgiven by SEQ ID NO: 31, the amino acid sequence of Balbc mouse BTLA isgiven by SEQ ID NO: 32, the amino acid sequence of C57BL6 is given bySEQ ID NO:33, and the amino acid sequence of cynomolgus monkey BTLA isgiven by SEQ ID NO: 34. The antibodies were then humanized and affinitymatured.

EXAMPLES

Expression and Purification of Engineered BTLA Agonist Antibodies

BTLA agonist antibodies of the present invention can be expressed andpurified essentially as follows. An appropriate host cell, such as HEK293 or CHO, can be either transiently or stably transfected with anexpression system for secreting antibodies using an optimalpredetermined HC:LC vector ratio (such as 1:3 or 1:2) or a single vectorsystem encoding both the HC and the LC. Clarified media, into which theantibody has been secreted, may be purified using any of manycommonly-used techniques. For example, the medium may be convenientlyapplied to a Mab Select column (GE Healthcare), or KappaSelect column(GE Healthcare) for Fab fragment, that has been equilibrated with acompatible buffer, such as phosphate buffered saline (pH 7.4). Thecolumn may be washed to remove nonspecific binding components. The boundantibody may be eluted, for example, by pH gradient (such as 20 mM Trisbuffer, pH 7.0 to 10 mM sodium citrate buffer, pH 3.0, or phosphatebuffered saline pH 7.4 to 100 mM glycine buffer, pH 3.0). Antibodyfractions may be detected, such as by SDS-PAGE, and then may be pooled.Further purification is optional, depending on intended use. Theantibody may be concentrated and or sterile filtered using commontechniques. Soluble aggregate and multimers may be effectively removedby common techniques, including size exclusion, hydrophobic interaction,ion exchange, multimodal, or hydroxyapatite chromatography. The purityof the antibody after these chromatography steps is between about 95% toabout 99%. The product may be held refrigerated, immediately frozen at−70° C., or may be lyophilized. Amino acid SEQ ID NOs for exemplifiedantibodies of the present invention are shown below.

TABLE 1 Amino acid sequences of exemplified BTLA agonist antibodies.Antibody SEQ ID NOs Antibody HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 22B3 1316 19 22 25 28 23C8 14 17 20 23 26 29 25F7 15 18 21 24 27 30Binding Affinity and Kinetics

The binding affinity and kinetics of the BTLA agonist antibodies of thepresent invention (22B3, 23C8, and 25F7) to BTLA are measured by surfaceplasmon resonance using Biacore® 3000 (GE Healthcare). The bindingaffinity is measured by immobilizing about 120 RU BTLA protein (human,rat, murine (Balbc or C57BL6), or cynomolgus monkey BTLA) via aminecoupling on a Biacore® CM5 chip, and flowing BTLA agonist antibody,starting from 500 nM in 2-fold serial dilution down to 15.6 nM. Theexperiments are carried out at 25° C. in HBS-EP buffer (GE HealthcareBR100669; 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, pH7.4). For each cycle, 250 μL antibody sample is flowed through flow cell1 and 2 at 50 μl/min, and then dissociated for 10 minutes. The chipsurface is regenerated with 5 μL injection of glycine buffer at pH 1.5at 10 μL/mL flow rate. The data are fit to a 1:1 Langmiur binding modelto derive k_(on), k_(off), and to calculate K_(D). Following proceduresessentially as described above, the following parameters (shown in Table2) were observed. Data shown below are the average of three experimentsfor human, cyno, rat and murine for 22B3.

TABLE 2 Binding affinity and kinetics. Antigen k_(on) k_(off) K_(D)Antibody (BTLA) (1/Ms) (1/s) (nM) 22B3 Human 5.87E+06 2.19E−03 0.365Cyno 2.45E+06 6.47E−04 0.27 Murine 2.60E+06 8.58E−02 32.5 (balbc) Murine1.89E+06 2.65E−01 147 (C57BL6) Rat 2.10E+06 4.62E−02 24.1 23C8 Human1.59E+05 2.93E−04  1.93 (n = 3) Cyno 8.71E+04 3.09E−03 35.35 (n = 2)Murine No Binding No Binding No Binding (balbc) Murine No Binding NoBinding No Binding (C57BL6) Rat Not Tested Not Tested Not Tested 25F7Human 6.8622E+04  1.42E−02 206.2 (n = 2) Cyno Not Tested Not Tested NotTested Murine Not Tested Not Tested Not Tested (balbc) Murine 7.70E+043.50E−04  4.63 (n = 1) (C57BL6) Rat Not Tested Not Tested Not Tested

As shown above in Table 2, the BTLA agonist antibodies of the presentinvention bind BTLA. Specifically, antibody 22B3 is able to bind human,murine, and cynomolgus monkey BTLA.

Binding to Primary Cells

The ability of BTLA antibodies of the present invention (22B3, 23C8, and25F7) to bind primary cells from different species is determined byFACS. Human peripheral blood mononuclear cells (PBMCs) are isolated froma donor blood sample (San Diego Blood Bank, # LRS-WBC) using Ficoll (GE#17-1440-02) and SepMate tubes (STEMCELL #15450), per manufacturer'sprotocol. Cyno PBMCs (WorldWide Primates # CA-10) are thawed from liquidnitrogen and washed once with FACS buffer (same as above).

Spleens from male C57BL6 mice (JAX) or female Sprague Dawley rats(Harlan) are harvested, pooled, and dissociated into single cellsuspensions using a cell strainer and syringe plunger over a 50 mLconical tube rinsed with RPMI 1640 complete with 10% heat inactivatedFBS and 2 mM EDTA. Cells are pelleted, media removed, and red bloodcells lysed by resuspending pellet in 2 ml ACK Lysing Buffer (gibco #A10492-01) for approximately 2 minutes before quenching with completeRPMI. Lysed cells may be pelleted and washed once in FACS buffer (DPBS1× containing 3% FBS, 20 mM HEPES, and 2 mM EDTA).

Isolated primary cells are quantified using a Countess cell counter, andresuspended at 2×10⁶ cells per ml in FACS buffer. Flow cytometryexperiments is performed the same day as cell isolation by plating 50 μl(˜0.1×10⁶) cells into a 96 well plate (Greiner #650101). Non-specificantibody binding is prevented by adding 1 μl Fc block (for example, fromBD #553142) for 15 minutes at 4° C. without washing.

BTLA antibody binding is tested at various concentrations, by serialdilution in FACS buffer. For example, a purified antibody and controlsstarting at different concentrations are first diluted to 30 μg/mL andserial 1:3 dilutions of the starting material is performed for a totalof 10 titrations (plus untreated control). Antibody titrations areincubated with cells for 20 minutes at 4° C., and washed with FACSbuffer prior to stain. Cells are stained using fluorochrome-conjugatedantibodies to identify specific cell types (eg. CD19 B cells, CD4 Tcells or CD8 T cells) or using a secondary antibody to identify thepresence or absence of antibody binding to that subset. Staining isperformed for 20 minutes at 4° C. and washed 3 times with FACS bufferprior to analysis on a flow cytometer. Results are analyzed usingstandard FACS analysis software (eg. FACSDiva) and reported as meanfluorescent intensity of the secondary antibody for each titration. Apositive result, which indicates binding, is determined by meanfluorescent intensity staining above background.

Following procedures essentially as described above, antibody 22B3 bindsto human, cynomolgus monkey, rat, and mouse BTLA-expressing cells,antibody 23C8 binds human and cynomolgus monkey BTLA-expressing cells,and antibody 25F7 binds human, cynomolgus monkey, and mouseBTLA-expressing cells.

BTLA Agonist Antibody-Induced Phosphorylation

To determine the ability of BTLA agonist antibodies of the presentinvention (22B3 and 25F7) to induce tyrosine phosphorylation in a humanB cell line, a BTLA antibody is bound to a 24-well culture plate at 10μg/mL for one hour at 37° C. hour. The plate is washed with PBS toremove any unbound antibody. A human BTLA-expressing B cell line, suchas Ramos.2G6.4C10 human B Lymphocyte cell line (ATCC), may be added tothe wells at 10×10{circumflex over ( )}6 cells/mL and incubated for 37°C. for 30 min. The cells are removed and lysed in Complete Lysis Buffer(MSD), and frozen at −80° C. for at least 30 min.

Phosphorylated-BTLA is detected by Meso Sector S 600. Streptavidindetection plates are prepared by incubating in blocking solution (MSD)for one hour at room temperature. A biotinylated-BTLA capture antibody(5A5) is coated onto the plate for one hour at room temperature followedby three or more Tris-wash steps. The cell lysates are incubated for twohours at room temperature. Total BTLA is detected with a SULFO-TAGanti-BTLA antibody (ANC6E9) and phosphorylated BTLA is measured with aSULFO-TAG anti-phosphotryosine antibody (PY20; MSD) followed by three ormore Tris-wash steps. Addition of 2× Read Buffer T (MSD) is then addedto the wells immediately prior to analysis using a Meso Sector S 600.

Following procedures essentially as described above, antibody 22B3resulted in a 2.41-fold increase in tyrosine phosphorylation of BTLAover background compared to negative control, and antibody 25F7 resultedin a 1.47-fold increase in tyrosine phosphorylation of BTLA overbackground compared to negative control. These data demonstrate that theBTLA agonist antibodies 22B3 and 25F7 are able to induce BTLAphosphorylation in a human B cell line.

Inhibition of Human Primary B Cell Proliferation

The in vitro potency of BTLA agonist antibodies of the present inventionare evaluated by the ability to inhibit human primary B cellproliferation. Human primary B cells are isolated from healthy humanperipheral blood mononuclear cells using human B cell isolation kit(EasySep) and are resuspended in appropriate human primary cell media.Anti-IgM is coated to plates along with titrations of isotype control orBTLA antibody and incubated for one hour at 37° C. followed by PBS washstep. Isolated human B cells are added to each well and incubated for 72hours at 37° C. with 5% CO2 followed by [³H]-thymidine pulse for thelast 18 hours. Post incubation plates are removed and placed on dry icefor 30 minutes and then stored at −20° C. until ready to harvest. Cellsare lysed by thawing and harvested with Harvester9600 (Tomtec).Proliferation is assessed by measuring [³H]-thymidine incorporation witha MicroBeta² 2450 Microplate Counter (Perkin Elmer).

Counts are used to assess relative proliferative response in this assay,and percent inhibition is calculated using the equation [%Inhibition=(AVGmaxsignal−signalsample)/AVGmaxsignal×100], which can beused to determine IC₅₀ values using graphing software (GraphPad Prism).

Following procedures essentially as described above, the BTLA agonistantibody 22B3 was able to inhibit primary B cell proliferation in vitrowith a calculated IC₅₀ of 0.32+/−0.1 nM, antibody 23C8 was able toinhibit primary B cell proliferation in vitro with a calculated IC₅₀ of0.14 nM, and antibody 25F7 was able to inhibit primary B cellproliferation in vitro with a calculated IC₅₀ of 0.17 nM. In a similarexperiment, antibody 22B3 was able to inhibit primary B cellproliferation with a calculated IC₅₀ of 0.32 nM, and an antibody havingthe same HCVRs and LCVRs as Mab8D5 (SEQ ID NO: 11 and 18 of the '694patent, respectively) inhibited primary B cell proliferation with acalculated IC₅₀ of 6.38 nM. These data demonstrate that the BTLA agonistantibodies 22B3, 23C8, and 25F7 are able to inhibit B cell proliferationin vitro, and that antibody 22B3 has greater in vitro activity ascompared to Mab8D5.

Humanized NSG Mouse Model of GvHD

Prevention of human PBMC-driven graft vs. host disease (GvHD) isdetermined in vivo.

Briefly, female NSG mice (JAX Labs, Stock #05557), approximately 8-10weeks old, are normalized and divided into treatment groups (n=8 miceper treatment group) based on baseline body weight measurements.Peripheral blood mononuclear cells (PBMCs) are isolated from a blooddonor program (San Diego Blood Bank, # LRS-WBC) using Ficoll (GE#17-1440-02) and SepMate tubes (STEMCELL #15450), per manufacturer'sprotocol. PBMCs are resuspended at approximately 150×106 cells per ml ofPBS. Treatment groups are blinded prior to dosing.

On day 1, 100 μl (15×106 cells) of PBMCs suspended in PBS (as describedabove) (or 100 μl PBS for non-engrafted controls) are injectedintravenously (IV) into the tail of each mouse. Mice are dosed weekly(QW) with antibody of the present invention (22B3 or 23C8) or controlsat varying concentrations in PBS vehicle, by subcutaneous (SQ)injections. Three independent studies are performed essentially asdescribed herein. Dosing concentrations for each study is [Study 1(antibody 22B3): 0.1, 1.0, 5.0, 10.0, and 20.0 mg/kg; Study 2 (antibody22B3 or 23C8): 0.001, 0.01, 10.0, and 100 mpk; and Study 3: 0.001,0.005, 0.01, 0.1, 0.5, and 1.0 mpk].

The study is terminated and mice are euthanized prior to isotype controlanimals losing 20% loss of baseline body weight (Studies 1 and 2) or day28 (Study 3). Weights are recorded (Study 1 and Study 2), serum iscollected for cytokine analysis (Study 1; analysis is performed by MSDELISA; cytokines analyzed are TNFα, IL-10, IL-6, IL-4, IL12p70, IL-13,IL-2, and IL-8), and spleens are harvested forphenotyping/pharmacodynamic analyses (measured by a reduction in CD 8 Tcell population; Study 1 and Study 3).

Following procedures essentially as described above, the following datawere obtained.

Antibody 22B3-treated animals in Study 1 demonstrated the following (atdoses 0.1, 1.0, 5.0, 10.0, or 20.0 mg/kg antibody): (i) similar bodyweights at the end of the study compared to the body weights ofnon-engrafted control animals; (ii) a reduction in the cytokines TNFα,IL-10, IL-6, IL-4, and IL-12p70 compared to isotype control animals; and(iii) a reduction in CD 8 T cell population compared to isotype controlanimals (phenotyping/pharmacodynamic analyses).

Data from Study 2 demonstrate that mice treated with 0.01 mg/kg antibody22B3, or 1.0, 5.0, or 10.0 mg/kg antibody 23C8 had similar body weightsat the end of the study compared to the body weights of non-engraftedcontrol animals. Study 2 did not demonstrate activity of 22B3 on bodyweight at 10.0 mg/kg, which may reflect natural donor variability ofthis model. In Study 3, antibody 22B3 demonstrated pharmacodynamicactivity in vivo at the following doses of antibody: 0.01, 0.1, 0.5, and1.0 mg/kg. Taken together, these data demonstrate that antibody 22B3 andantibody 23C8 were efficacious in preventing GvHD in vivo.

mIFNα-Induced Lupus Nephritis

The interferon-alpha (IFNα)-induced lupus nephritis model is a mousemodel of systemic lupus erythematosus (SLE) in which IFNα is used tosynchronize onset and accelerate progression of disease in a cross withNew Zealand Black and New Zealand White (NZB/W) mice. The NZB/W mousemodel is a classical model of spontaneous lupus nephritis. The diseaseprogression in these mice could be accelerated with exogenousadministration of IFNα using adenovirus vectors. This lupus nephritismodel is used to demonstrate the activity of the BTLA agonist antibodiesof the present invention.

One day before the study start, eleven week old female NZB/W mice arerandomly sorted based on body weight. Mice are distributed into thefollowing treatment groups: (1) LacZ adeno-associated virus (AAV+10mg/kg human IgG4 PAA isotype control (PAA is S228P, F234A, and L235Amutations), (2) IFNα AAV+10 mg/kg human IgG4 PAA isotype control, (3)IFNα AAV+3 mg/kg 22B3 antibody, (4) IFNα AAV+10 mg/kg 22B3 antibody, or(5) IFNα AAV+50 mg/kg cyclophosphamide. On study start date (Day 0),mice are either administered once with 10¹¹ genome copies (GC) of AAVexpressing LacZ gene (non-diseased) or mouse IFNα (diseased) in PBSintravenously. In groups 1-4, the mice are treated with isotype controlor 22B3 antibody antibodies in PBS subcutaneously once every weekstarting on Day 0. In group 5, mice are treated with cyclophosphamide inPBS intraperitoneally every 10 days. Urine samples are collected fromthe mice every 2 weeks until study termination 6 weeks after treatmentinitiation. The Kamiya Biomedical™ mouse microalbumin ELISA is used toquantitate urine albumin levels. Urine creatinine is measured by usingan enzymatic creatinine assay (Roche Diagnostics). Albuminuria, abiomarker of renal function, is defined as greater than 300 μg albuminper mg creatinine detected in the urine.

Following procedures essentially as described above, by week 4, theincidence of albuminuria in the isotype treated diseased group (IFNαAAV+hIgG4 PAA) reached 100% and stayed elevated until end of study,while the LacZ AAV treated (non-diseased) mice did not show anyincidence of albuminuria. Cyclophosphamide, which can be acutelynephrotoxic, caused a transient increase in albuminuria in diseasedmice, but the incidence of albuminuria in the cyclophosphamide group wasreduced to zero by study end. Antibody 22B3 at 3 mg/kg and 10 mg/kg wasable to reduce incidence of albuminuria to 50% and 20%, respectively, atday 28, and 60% and 70%, respectively, at day 42. These resultsindicated that antibody 22B3 was able to preserve renal function in themodel.

A Kaplan-Meier plot (data not shown) of percent survival during thestudy showed that renal insufficiency in the isotype treated diseasedgroup led to deaths starting at day 28. By the end of the study,survival rate in the isotype treated diseased group was 60%. Thenon-diseased and cyclophosphamide treated groups had survival rates of100%. The mice treated with 10 mg/kg antibody 22B3 also showed 100%survival at the end of the study, while the mice treated with 3 mg/kgshowed 80% survival. These results indicated that antibody 22B3 was ableto prevent disease related deaths in this model.

Imiquimod-Induced Model of Psoriasis

The ability of an antibody of the present invention to limit theseverity of psoriasis-like dermatitis induced by application of theTLR7/8 agonist imiquimod (IMQ) is tested. Seven-week-old, femaleB6.SJL-Ptprc^(a) Pepc^(b)/BoyJ mice (JAX stock number: 002014), orHVEM^(−/−) mice (described in Wang et al, J. Clin. Invest., 115:3,711-717, March 2005) are injected intraperitoneally with 3 mg/kg or 1mg/kg of antibody 22B3 or antibody 25F7, respectively, on day 0, and thebacks of the mice are shaved. Animals injected with hIgG4 isotypecontrol served as controls. On days 1-3, mice are anesthetized withinhaled isoflurane (VetOne), and 5% IMQ cream (50 mg, Fougera) is thenapplied to a defined area of the shaved skin. On day 4, the treated areaof skin is excised and analyzed for disease severity andinflammation-related gene expression.

Following procedures essentially as described above, histologicalanalysis demonstrated thickening of the epidermal layer withparakeratosis and hyperkeratosis in the groups treated with hIgG4isotype control or 1 mg/kg antibody 22B3. Mice treated with 3 mg/kgantibody 22B3 or 3 mg/kg antibody 25F7 showed a significant reduction inepidermal thickness, with some areas appearing histologically normal.Gene expression in the skin was analyzed by qPCR using the iTaqUniversal SYBR Green Supermix (Bio-Rad). Mice treated with 3 mg/kgantibody 22B3 exhibited a significant decrease in expression of Type IIFNs (IFNα, IFNβ) and IFNγ, as well as IFN-response genes (Isg15, Mx1,Mx2, Oas2). Analysis of cytokines involved in establishing IMQ-induceddermatitis also demonstrated a significant reduction in IL-22 and IL-23expression in the 3 mg/kg antibody 22B3 treatment group. These datademonstrate that the BTLA agonist antibodies 22B3 and 25F7 are able toreduce epidermal thickness in a mouse model of psoriasis-likedermatitis.

Epitope Determination

The functional epitopes of the BTLA agonist antibodies of the presentinvention are determined by ELISA, and the structural epitopes aredetermined by x-ray crystallography.

Methods ELISA: Functional Epitope

The following set of surface mutations of BTLA were introducedindividually into a human BTLA protein fused to (human) Fc: D35R, Q37R,Y39E, R42D, Q43A, E45R, S47H, L49R, D52R, E55R, E57R, D84R, N65R, H68A,V80R, K81E, E83R, S88H, K90H, E91H, I95R, E103H, L106R, N108R, R114V,S121Y, N122R, E125H, H127E, T130R, Y132R, and T134H.

Binding of 22B3 and 23C8 was determined using an ELISA wherein theantibody to be epitope mapped was captured by an immobilized anti-rabbitantibody and after washing each BTLA mutant was incubated as a 4 point4-fold dilution series with the captured antibody and detected with anenzyme linked anti-human Fc reagent. The resulting signal was comparedamong antibodies and to control antibodies. The functional epitopenormally indicated itself by a dramatic reduction in signal for one ortwo mutants. For the 25F7 antibody, a sandwich ELISA was performed,wherein humanized 22B3 was immobilized, BTLA mutants were captured, andbound by rabbit 25F7. This gave a much stronger signal and the 25F7epitope could be identified after eliminating the 22B3 epitope.

X-Ray Crystallography: Structural Epitope

In order to determine interacting interfaces and therefore the physicalepitope on BTLA of the various antibodies, human BTLA wasco-crystallized with the Fab portion of an antibody of the presentinvention and a crystal structure was determined. From the resultingcrystal structure, the BTLA residues within 4.5A of any antibody atomwere counted as part of the epitope (using the Pymol visualizationsoftware). 4.5 angstroms is measured from atom center to atom center.Any residue with at least one atom that is 4.5 angstroms close to anyatom in the antibody is part of the epitope.

Two 22B3 structures were determined in complex with human BTLA. Thefirst utilized the parent rabbit 22B3 antibody Fab, Histidine tagged andpurified with a S47H mutant (stabilizing mutation) of human BTLAexpressed as an Fc fusion and then cleaved and purified. These twoproteins were mixed at an approximately equimolar ratio and screened incommercially available screens for crystallization. Crystals wereobtained and diffraction data collected at the Advanced Photon Source.This data was reduced and solved by molecular replacement and refined toyield a high resolution structure of the complex between 22B3 and BTLA.The second complex was between an affinity matured version (with HCmutations I56Q/T57H/G98A and LC S95H) of the humanized 22B3 (Fabportion) and human BTLA. These were co-expressed, purified as a complexand similarly screened. The resulting structure and epitope were similarto the first structure.

The structure of 23C8 in complex was obtained in the same way as thefirst 22B3 complex, namely by purifying the His tagged rabbit parentFab, mixing with monomeric S47H human BTLA and crystallizing.

The structure of 25F7 in complex with human BTLA was obtained as per thesecond 22B3 complex, namely by co-expression, co-purification andcrystallization. A double mutant of the humanized 25F7 with improvedbinding to human BTLA was utilized (humanized 25F7 used for epitopedetermination has mutations at HC S30W/LC E27R).

Results

22B3 antibody: Among a set of BTLA surface mutants, R42D and H127E had asignificant negative impact on binding to rabbit 22B3 antibody(comprising the same CDRs as 22B3 but with a rabbit framework). Thefunctional epitope comprises Arg at position 42 and His at position 127of human BTLA (SEQ ID NO:31). BTLA residues that are within 4.5angstroms of 22B3 in the crystal structure complex between human BTLAand rabbit 22B3 Fab, and are the structural epitope, are the followingresidues of SEQ ID NO: 31: Asp at position 35, Gln at position 37 to Argat position 42, Leu at position 74, Gly at position 76 to Cys atposition 79, Arg at position 114, Phe at position 119, Gln at position120, and Asn at position 122 to Ser at position 128. BTLA residues thatare within 4.5 angstroms of 22B3 in the crystal structure complexbetween human BTLA and a human 22B3 variant (HC I56Q/T57H/G98A LC S95H)Fab are Asp at position 35, Gln at position 37 to Arg at position 42,Leu at position 74, Gly at position 76 to Cys at position 79, Arg atposition 114, Phe at position 119, Gln at position 120, Asn at position122, and Ile at position 124 to Ser at position 128 of SEQ ID NO:31.

In a similar study, the structural epitope for HVEM binding BTLA was thefollowing amino acids of BTLA: Gln at position 37 to Arg at position 42,Leu at position 74, Gly at position 76, Thr at position 77, Ser atposition 112, Arg at position 114, Asn at position 118, Ser at position121 to Ser at position 128, and Thr at position 130. Structuralsimilarity between antibody 22B3 and HVEM was assessed by superimposingthe antibody:BTLA crystal structure onto the HVEM:BTLA crystal structurealigning the BTLA molecules. The backbone root-mean-square deviation inthe HVEM region containing amino acid residues 69-72 and thecorresponding antibody region was determined to be 1.4 angstroms.

23C8 antibody: D52R blocks binding of rabbit 23C8 (comprising the sameHCDR1, HCDR2, HCDR3, LCDR1 and LCDR2 as 23C8, having the LCDR3 ofQCTYGGVVGSTSDDNP, and having a rabbit framework) to human BTLA in anELISA. The functional epitope comprises Asp at position 52 of human BTLA(SEQ ID NO:31). BTLA residues that are within 4.5 angstroms of 23C8 inthe crystal structure complex between human BTLA (S47H) and rabbit 23C8Fab, and are the structural epitope, are His at position 46 to Glu atposition 55, Glu at position 103, Pro at position 104, Leu at position106, Pro at position 107, Thr at position 134 to Ala at position 139 ofSEQ ID NO:31. Antibody 23C8 does not mimic HVEM binding.

25F7 antibody: Among a set of BTLA surface mutants, H68A and K61E had asignificant negative impact on binding to rabbit 25F7 antibody(comprising the same CDRs as 25F7 but with a rabbit framework). Thefunctional epitope comprises His at position 68, and Lys at position 81,of human BTLA (SEQ ID NO:31). BTLA residues that are within 4.5angstroms of 25F7 in the crystal structure complex between human BTLAand humanized 25F7 Fab variant (HC S30W, LC E27R), and are thestructural epitope, are Tyr at position 62, Ala at position 64 to His atposition 68, Arg at position 85 to Glu at position 91, Phe at position98, and Asn at position 118 of SEQ ID NO:31. Antibody 25F7 does notmimic HVEM binding.

Sequences HC of Antibody 22B3 (SEQ ID NO: 1)QVQLVQSGAEVKKPGASVKVSCKASGFSLSSYGVSWVRQAPGQGLEWMGAISYDGITYYASWAKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDYYDDYVYVYALDIWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGLC of Antibody 22B3 (SEQ ID NO: 2)EIVLTQSPGTLSLSPGERATLSCQASQSISTALAWYQQKPGQAPRLLIYAASTLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGYSSSNLDNVFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECHCVR of Antibody 22B3 (SEQ ID NO: 3)QVQLVQSGAEVKKPGASVKVSCKASGFSLSSYGVSWVRQAPGQGLEWMGAISYDGITYYASWAKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDYYDDYVY VYALDIWGQGTLVTVSSLCVR of Antibody 22B3 (SEQ ID NO: 4)EIVLTQSPGTLSLSPGERATLSCQASQSISTALAWYQQKPGQAPRLLIYAASTLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGYSSSNLDNVFGGGTKVEIKHC of Antibody 23C8 (SEQ ID NO: 5)EVQLVESGGGLVQPGGSLRLSCAASGFDISKYNIQWVRQAPGKGLEWVGFINYGGSAYYASRAKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARGLSNSDLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCVMHEALHNHYTQKSL SLSLGLC of Antibody 23C8 (SEQ ID NO: 6)DIQMTQSPSSLSASVGDRVTITCQASQSISSWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQSTYGGVVGSTSDDNPFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECHCVR of Antibody 23C8 (SEQ ID NO: 7)EVQLVESGGGLVQPGGSLRLSCAASGEDISKYNIQWVRQAPGKGLEWVGFINYGGSAYYASRAKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARGLSNSDLWGQ GTLVTVSSLCVR of Antibody 23C8 (SEQ ID NO: 8)DIQMTQSPSSLSASVGDRVTITCQASQSISSWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQSTYGGVVGSTSDDNPFGGGTKVEI KHC of Antibody 25F7 (SEQ ID NO: 9)QVQLVQSGAEVKKPGASVKVSCKASGFSLSTYAMNWRQAPGQGLEWMGIISDDGTTYYATWAKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDAGAGGVQDYLTLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGLC of Antibody 25F7 (SEQ ID NO: 10)DIVMTQSPDSLAVSLGERATINCQASENIYNFLAWYQQKPGQPPKLLIYSASTLASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQGSSNSNIDNPFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECHCVR of Antibody 25F7 (SEQ ID NO: 11)QVQLVQSGAEVKKPGASVKVSCKASGFSLSTYAMNWVRQAPGQGLEWMGIISDDGTTYYATWAKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDAGAGGVQ DYLTLWGQGTLVTVSSLCVR of Antibody 25F7 (SEQ ID NO: 12)DIVMTQSPDSLAVSLGERATINCQASENIYNFLAWYQQKPGQPPKLLIYSASTLASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQGSSNSNIDNPFGGGTKVEIKHCDR1 of Antibody 22B3 (SEQ ID NO: 13) GIFSILSSYGVSHCDR1 of Antibody 23C8 (SEQ ID NO: 14) GFDISKYNIQHCDR1 of Antibody 25F7 (SEQ ID NO: 15) GFSLSTYAMNHCDR2 of Antibody 22B3 (SEQ ID NO: 16) AISYDGITYYASWAKSHCDR2 of Antibody 23C8 (SEQ ID NO: 17) FINYGGSAYYASRAKGHCDR2 of Antibody 25F7 (SEQ ID NO: 18) IISDDGTTYYATWAKGHCDR3 of Antibody 22B3 (SEQ ID NO: 19) GDYYDDYVYVYALDIHCDR3 of Antibody 23C8 (SEQ ID NO: 20) GLSNSDLHCDR3 of Antibody 25F7 (SEQ ID NO: 21) DAGAGGVQDYLTLLCDR1 of Antibody 22B3 (SEQ ID NO: 22) QASQSISTALALCDR1 of Antibody 23C8 (SEQ ID NO: 23) QASQSISSWLSLCDR1 of Antibody 25F7 (SEQ ID NO: 24) QASENIYNFLALCDR2 of Antibody 22B3 (SEQ ID NO: 25) AASTLASLCDR2 of Antibody 23C8 (SEQ ID NO: 26) RASTLASLCDR2 of Antibody 25F7 (SEQ ID NO: 27) SASTLASLCDR3 of Antibody 22B3 (SEQ ID NO: 28) QQGYSSSNLDNVLCDR3 of Antibody 23C8 (SEQ ID NO: 29) QSTYGGVVGSTSDDNPLCDR3 of Antibody 25F7 (SEQ ID NO: 30) QQGSSNSNIDNPHuman BTLA (SEQ ID NO: 31)MKTLPANILGTGKLFWVFFLIPYLDIWNIFIGKESCDVQLYIKRQSEHSILAGDPFELECPVKYCANRPHVTWCKLNGTTCVKLEDRQTSWKEEKNISFFILHFEPVLPNDNGSYRCSANFQSNLIESHSTTLYVTDVKSASERPSKDEMASRPWLLYRLLPLGGLPLLITTCFCLFCCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEA PTEYASICVRSMouse Balbc BTLA (SEQ ID NO: 32)MKTVPANILGTPRLFREFFILHLGLWSILCEKATKRNDEECEVQLNIKRNSKHSAWTGELFKIECPVKYCVHRPNVTWCKHNGTIWVPLEVGPQLYTSWEENRSVPVFVLHFKPIHLSDNGSYSCSTNFNSQVINSHSVTIHVRERTQNSSEHPLITVSDIPDATNASGPSTMEERPGRTWLLYTLLPLGALLLLLACVCLLCFLKRIQGKEKKPSDLAGRDTNLVDIPASSRTNHQALPSGTGIYDNDPWSSMQDESELTISLQSERNNQGIVYASLNHCVIGRNPRQENNMQEAPTEYASICVRS Mouse C57BL6 BTLA (SEQ ID NO: 33)MKTVPAMLGTPRLFREFFILHLGLWSILCEKATKRNDEECPVQLTITRNSKQSARTGELFKIQCPVKYCVHRPNVTWCKHNGTICVPLEVSPQLYTSWEENQSVPVFVLHFKPIHLSDNGSYSCSTNFNSQVINSHSVTIHVTERTQNSSEHPLITVSDIPDATNASGPSTMEERPGRTWLLYTLLPLGALLLLLACVCLLCFLKRIQGKEKKPSDLAGRDTNLVDIPASSRTNHQALPSGTGIYDNDPWSSMQDESELTISLQSERNNQGIVYASLNHCVIGRNPRQENNMQEAPTEYASICVRSCynomolgus Monkey BTLA (SEQ ID NO: 34)MKTLPAMLGSGRLFWVVFLIPYLDIWNIHGKESCDVQLYIKRQSYHSIFAGDPFKLECPVKYCAHRPQVTWCKLNGTTCVKLEGRHTSWKQEKNLSFFILHFEPVLPSDNGSYRCSANFLSAIIESHSTTLYVTDVKSASERPSKDEMASRPWLLYSLLPLGGLPLLITTCFCLFCFLRRHQGKQNELSDTTGREITLVDVPFKSEQTEASTRQNSQVLLSETGIYDNEPDFCFRMQEGSEVYSNPCLEENKPGIIYASLNHSIIGLNSRQARNVKEA PTEYASICVRSExemplified DNA for Expressing Antibody 22B3 Heavy Chain of SEQ ID NO: 1(SEQ ID NO: 35)caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtttcctgcaaggcatctggattctccctcagtagctatggagtgagctgggtgcgacaggcccctggacaagggcttgagtggatgggagccattagttatgatggtattacatactacgcgagctgggcgaaaagcagagtcaccatgaccagggacacgtccacgagcacagtctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagaggggactactacgatgattatgtttatgtttatgctttagacatctggggccagggcaccctggtcaccgtctcctcagcttctaccaagggcccatcggtcttcccgctagcgccctgctccaggagcacctccgagagcacagccgccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacgaagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtcccccatgcccaccctgcccagcacctgaggccgccgggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggaaagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctctgggtExemplified DNA for Expressing Antibody 22B3 Light Chain of SEQ ID NO: 2 (SEQID NO: 36)GaaattgtgttgacgcagtaccaggcaccagtattgtaccaggggaaagagccaccactcctgccaggccagtcagagcattagtactgcattagcctggtaccagcagaaacctggccaggctcccaggctcctcatctatgctgcatccactaggcatctggcatcccagacaggttcagtggcagtgggtagggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcaacagggttatagtagtagtaatcttgataatgttttcggcggagggaccaaggtggagatcaaacggaccgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctagttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccaccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgcExemplified DNA for Expressing Antibody 23C8 Heavy Chain of SEQ ID NO: 5(SEQ ID NO: 37)gaggtgcagaggtggagtagggggaggcttggtccagcctggagggtccctgagactacctgtgcagcctaggattcgacatcagtaagtacaacatccaatgggtccgccaggctccagggaaggggctggagtgggttggcttcattaattatggtggtagcgcatactacgcgagccgggcgaaaggcagattcaccatctcaagagatgattcaaagaactcactgtatctgcaaatgaacagcctgaaaaccgaggacacggccgtgtattactgtgctagaggactaagtaatagcgacctaggggccagggcaccaggtcaccgtctcctcagcttctaccaagggcccatcggtcttcccgctagcgccctgaccaggagcacctccgagagcacagccgccagggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccaccagcagcttgggcacgaagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtcccccatgcccaccagcccagcacctgaggccgccgggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtaccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccagcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggaaagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgaccgtgatgcatgaggactgcacaaccactacacacagaagagcctaccagtactgggtExemplified DNA for Expressing Antibody 23C8 Light Chain of SEQ ID NO: 6 (SEQID NO: 38)gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccaggccagtcagagcattagtagttggttatcctggtatcagcagaaaccagggaaagcccctaagctcctgatctacagggcatccactctggcatctggggtcccatcaaggttcagtggaagtggatctgggacagattttactttcaccatcagcagcctgcagcctgaagatattgcaacatattactgtcaatccacttatggtggtgttgttggcagtactagtgatgataatcctttcggcggagggaccaaggtggagatcaaacggaccgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgcExemplified DNA for Expressing Antibody 25F7 Heavy Chain of SEQ ID NO: 9(SEQ ID NO: 39)caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtttcctgcaaggcatctggattctccctcagtacctatgcaatgaactgggtgcgacaggcccctggacaagggcttgagtggatgggaatcattagtgatgatggtaccacatactacgcgacctgggcgaaaggcagagtcaccatgaccagggacacgtccacgagcacagtctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagagatgctggtgctggtggtgtccaagactacttaaccttgtggggccagggcaccctggtcaccgtctcctcagcttctaccaagggcccatcggtcttcccgctagcgccctgctccaggagcacctccgagagcacagccgccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacgaagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtcccccatgcccaccctgcccagcacctgaggccgccgggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggaaagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctctgggtExemplified DNA for Expressing Antibody 25F7 Light Chain of SEQ ID NO: 10(SEQ ID NO: 40)GacatcgtgatgacccagtaccagactccctggctgtgtactgggcgagagggccaccatcaactgccaggccagtgagaatatttacaactttttggcctggtaccagcagaaaccaggacagcctcctaagagctcatttactagcatccactaggcatctggggtccctgaccgattcagtggcagcgggtagggacagatttcactctcaccatcagcagcctgcaggctgaagatgtggcagtttattactgtcaacagggttctagtaatagtaatattgataatcctttcggcggagggaccaaggtggagatcaaacggaccgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctagttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccaccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgcHuman HVEM (SEQ ID NO: 41)MEPPGDWGPPPWRSTPKTDVLRLVLYLTFLGAPCYAPALPSCKEDEYPVGSECCPKCSPGYRVKEACGELTGTVCEPCPPGTYIAHLNGLSKCLQCQMCDPAMGLRASRNCSRTENAVCGCSPGHFCIVQDGDHCAACRAYATSSPGQRVQKGGTESQDTLCQNCPPGTFSPNGTLEECQHQTKCSWLVTKAGAGTSSSHWVWWFLSGSLVIVIVCSTVGLIICVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIP SFTGRSPNH

We claim:
 1. An antibody that binds BTLA, comprising HCDR1 having theamino acid sequence of SEQ ID NO: 13, HCDR2 having the amino acidsequence of SEQ ID NO: 16, HCDR3 having the amino acid sequence of SEQID NO: 19, LCDR1 having the amino acid sequence of SEQ ID NO: 22, LCDR2having the amino acid sequence of SEQ ID NO: 25, and LCDR3 having theamino acid sequence of SEQ ID NO:
 28. 2. The antibody of claim 1,comprising a heavy chain variable region (HCVR) having the amino acidsequence of SEQ ID NO: 3, and a light chain variable region (LCVR)having the amino acid sequence of SEQ ID NO:
 4. 3. The antibody of claim1, comprising a heavy chain (HC) having the amino acid sequence of SEQID NO: 1, and a light chain (LC) having the amino acid sequence of SEQID NO:
 2. 4. The antibody of claim 1, comprising two HC and two LC,wherein each HC has the amino acid sequence of SEQ ID NO: 1, and eachlight chain (LC) has the amino acid sequence of SEQ ID NO:
 2. 5. Apharmaceutical composition comprising the antibody of claim 1, and oneor more pharmaceutically acceptable carriers, diluents, or excipients.6. An antibody obtainable by a) cultivating a mammalian cell comprisinga DNA molecule comprising a polynucleotide sequence that encodes theantibody heavy chain having the amino acid sequence of SEQ ID NO: 1 anda polynucleotide sequence that encodes the antibody light chain havingthe amino acid sequence of SEQ ID NO: 2 under conditions such that theantibody is expressed, and b) recovering the expressed antibody.
 7. Amethod of treating a patient having a rheumatic disease selected from atleast one of lupus nephritis, systemic lupus erythematosus, andrheumatoid arthritis, comprising administering to the patient aneffective amount of an antibody of claim
 1. 8. A method of treating apatient having a dermatology disease selected from at least one ofatopic dermatitis and psoriasis, comprising administering to the patientan effective amount of an antibody of claim
 1. 9. A method of treating apatient having multiple sclerosis comprising administering to thepatient an effective amount of an antibody of claim 1.